The lead-target interaction is optimized with respect to increasing affinity, reducing steric hindrance and generating specificity. These optimizations are carried out primarily through site-directed or random mutagenesis of the parental plasmid. The mutant plasmids are typically transformed into a host to generate enough plasmid to transform or transfect into an expression system. Once expressed, the mutant proteins should be purified for subsequent in vitro assays. Failure to do so introduces myriad variables from crude lysates that can mean false positives, false negatives and artifacts. Purification of the large number of mutant leads is daunting since there are many iterations of the drug lead in order to optimize the affinity, specificity and half-life. With so many mutants designed, expressed and tested, lead optimization is naturally a good candidate for high throughput automation.
Functional screens often performed on crude lysates. Use PhyTip columns for cleanup to remove artifacts, false positives and false negatives.
Protein Purification Applications
- Affinity Pull Down
- Immunoprecipitation / Co-immunoprecipitation
- Chromatin Immunoprecipitation
- Protein-Antibody Engineering
- Target Discovery
- DNA Encoded Library Screening
- Pharmacokinetic / Pharmacodynamic Modeling
- Quality Control
- Library Screening (Fabs, scFvs, small molecules)
- X-ray Crystallography, Membrane Proteins
- Lead Optimization / Screening
- Expression Optimization / Screening
- Epitope Mapping